Our studies of the CRTAP gene, encoding cartilage associated protein, and the discovery that its mutations cause recessive osteogenesis imperfecta (OI) have led to the identification of additional non-collagen gene mutations responsible for this disease. Most of these mutations occur in rER resident proteins that are involved in fibrillar collagen modification, folding and assembly. Importantly, while some of these processes are relatively well understood others, such as prolyl 3-hydroxylation, have remained quite unexplored and deserve more attention. Because prolyl 3-hydroxylation is found in various types of collagens, both fibrillar and non- fibrillar, the identification of the molecular componens responsible for this modification and how they work together in different tissues will provide important clues about tissue-specific matrix molecules production and novel mechanisms of disease. Based on previous characterization of the prolyl 3-hydroxylation complex, containing Crtap, P3h1 and CypB, we now propose to determine the role of Sc65, a novel member of the Leprecan gene family and the most closely related protein to Crtap. Our preliminary data show that Sc65 is a rER protein, highly expressed in the skeleton and when mutated causes a low bone mass phenotype. We hypothesize that Sc65 mediates interactions with the other prolyl 3-hydroxylase enzymes, P3h2 and/or P3h3, contributes to collagen post-translational modification at sites that are distinct from those modified by the Crtap complex and, when mutated, causes the development of OI. To test these hypotheses, the following specific aims are proposed: 1) Comprehensively characterize the skeletal phenotype of mice with a homozygous gene- trap insertion in the Sc65 gene using micro-CT, bone histomorphometry, collagen mass spectrometry and FTIRI; 2) Determine effects of Sc65 mutation on bone cell differentiation using primary calvarial osteoblasts and ex-vivo bone-marrow osteogenic and osteoclastogenic cultures from Sc65 mutant and WT control mice; 3) Determine the sub-cellular localization of the Sc65 protein and identify its interacting partners by co- immunoprecipitation and protein sequencing; and 4) Generate a conditional knock-out mouse by creation of a Sc65 floxed allele, inactivate it in bone and characterize its skeletal phenotype. Successful completion of this work will 1) elucidate a novel role for Sc65 in skeletal development and bone homeostasis, 2) identify Sc65 protein interactors which will provide insight into the molecular mechanisms involved in collagen prolyl 3- hydroxylation, 3) enhance understanding of the Leprecan genes in skeletal development and other tissues, and 4) provide further insight into the role of these non-collagenous proteins in the etiology of human connective tissue disease, including OI. PUBLIC HEALTH RELEVANCE: Role of Leprecan genes in skeletal formation The Leprecan family of genes, including Crtap, P3h1, P3h2, P3h3 and Sc65, has been recently identified and mutations in some of its members (Crtap and P3h1) are associated with forms of the human disease known as osteogenesis imperfecta (OI). Among Leprecans, Sc65 is probably the least characterized although it bears the highest similarity to Crtap and likely represents a paralog gene. Our preliminary data indicate that it encodes an ER protein and that, like Crtap, it has an important function during bone formation. We will investigate its role during skeletal formation and doing so will gain additional insights into the function of Crtap and of collagen prolyl 3-hydroxylation.